Found in various eukaryotic organisms, polo-like kinases (collectively, Plks) are a conserved subfamily of Ser/Thr protein kinases that play critical roles in cell proliferation. Plks are characterized by the presence of a highly conserved C-terminal polo-box domain (PBD) composed of two structurally-related PB1 (residues 411-489 in Plk1) and PB2 (residues 511-592) motifs. Multiple forms of Plks, designated Plk1, Plk2/Snk, Plk3/Prk/Fnk, and Plk4/Sak, exist in mammals. Plk4 is the most distantly related member of the Plk subfamily and one of the two Plk4 variants, Sak-a, contains only the PB1 motif near the end of an unusually long C-terminal extension. Among the Plks, Plk1 has been studied most extensively because of its ability to override cellular checkpoints and induce genetic instability, leading to oncogenic transformation of human cells. Not surprisingly, Plk1 is overexpressed in a broad spectrum of human cancers and has been proposed as a new prognostic marker for many types of malignancies.
Furthermore, interference with Plk1 function induces apoptotic cell death in most tumor cells, but not in normal cells, and reduces tumor growth in mouse xenograft models. A Plk1 inhibitor known as BI 6727 (volasertib) is presently undergoing clinical trials for the treatment of various human cancers, including acute myeloid leukemia. In contrast to the role of Plk1 in cell proliferation and tumorigenesis, the two most closely related kinases, Plk2 and Plk3, appear to play a role in checkpoint-mediated cell cycle arrest to ensure genetic stability and prevent oncogenic transformation. Thus, specific inhibition of Plk1, but not Plk2 or Plk3, is critically important for anti-Plk1 cancer therapy.
The PBD of Plk1 plays a critical role in proper subcellular localization and mitotic functions of Plk1 by interacting with phosphorylated Ser/Thr peptides with the invariable Ser residue at the −1 position (S-p-S/T motif). Crystal structures of the Plk1 PBD in complex with artificial phosphopeptides optimized for PBD binding have revealed that the PB1 and PB2 motifs have identical folds described as β6α (a six-stranded anti-parallel β-sheet and an α-helix) and form a hetero-dimeric phosphopeptide-binding module.
The phosphopeptide binds to a cleft formed between PB1 and PB2 and interacts with key amino acid residues from both polo-boxes. His538 and Lys540 from PB2 are pivotal for electrostatic interactions with the negatively charged phosphate group of phospho-Ser/Thr (p-Ser/Thr) residue, whereas Trp414 from PB1 is critical for the selection of Ser at the −1 position by engaging in two hydrogen bonding interactions and van der Waals interactions with the Ser-1 residue. These residues are conserved in the PBDs of Plk1, Plk2, and Plk3 (in short, Plk1-3), attesting to their importance (Plk4 has a distinct binding module and forms a homodimer through a motif called cryptic polo-box).
By examining PBD-binding phosphopeptides, the phosphopeptide “PLHSpT” was identified that specifically interacts with the Plk1 PBD with a high affinity, but not with the two closely-related Plk2 and Plk3 PBDs. Based on this peptide sequence, peptides with high PBD-binding affinity may be designed and prepared; however, even with high PBD-binding affinity, it is difficult for the peptides to achieve activity in whole-cell systems, possibly due to poor bioavailability arising from poor solubility or limited membrane transport (or both). There is a need in the art to design and prepare PBD-binding peptides with improved pharmaceutical properties, including increased bioavailability.